Nerve Growth Factor (NGF) is a key differentiation and maintenance protein for sensory, sympathetic, and central neurons. Information is available about the protein structure of the active beta-subunit and the 7S oligomeric form of the protein, the developmental role of NGF in the nervous system, the structure of its plasma membrane receptor, the cellular dynamics and movement of the receptor subsequent to binding, and some aspects of the signalling required for differentiation of responsive cells. Information about the structure-function relationships of the beta-NGF molecule is essential for understanding such neuronotrophic proteins. The overall goal of this project is to determine structural features of the active beta-NGF subunit that are responsible for interaction with the cellular receptor and resultant intracellular signalling, using recombinant DNA and immunochemical approaches. Molecular engineering.
One aim i s to obtain beta-NGF variants with amino acid replacements in four selected regions of the molecule and correlate these structural changes with biological properties. In the previous grant period biologically active NGF has been successfully expressed in an E. coli system utilizing the cloned moused cDNA. Site specific mutagenesis of critical amino acids will now allow determination of the role of individual residues in biological function. Antagonists and partial agonists of beta-NGF may also be obtained for biological studies. Immunochemistry. Another aim is to determine surface epitopes of the NGF molecule involved in receptor recognition by producing site-specific antibodies directed against synthetic oligopeptides of the NGF protein. Oligopeptides corresponding to particular sequences in the beta-NGF molecule are being synthesized, site specific antibodies toward them will be produced, and the antibodies will be used to ascertain the functional role of surface portions of the beta-NGF molecule. These mutant NGF proteins and site specific antibodies will be studied by neurite outgrowth in rat Pheochromocytoma (PC12) cells and chick dorsal root sensory neurons, in receptor binding and cellular trafficking in PC12 cells, in cellular signalling and gene regulation, in the self-dimerization of beta-NGF, in the formation of the 7S oligomer, and analysis of the conformation of the recombinant protein. The results of these studies will further our knowledge of the fundamental structural requirements for the interaction of NGF with neuronal targets and elucidate molecular and cellular aspects of neuronal differentiation and survival. Novel analogues of NGF will be produced that should be useful in developmental studies. this information will eventually lead to better understanding of neurodevelopmental problems and related diseased such as Alzheimer's and Huntington's diseases.
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